Evaluation of the Cytotoxicity of Two Types of Triple Antibiotic Paste on Human Permanent Dental Apical Papilla Stem Cells: an in vitroin vitro Study

Statement of the Problem: The use of a new antimicrobial combination in the regenerative endodontic treatment of immature teeth pulp necrosis is a well-known method. Concerns have been raised about the destructive effect of this combination on the stem cells from the apical papilla of permanent human teeth, and there is a study gap. Purpose: The main objective of the present study was to investigate the cytotoxic effect of modified triple antibiotic paste (mTAP) on stem cells from the apical papilla (SCAPs) of permanent human teeth. Materials and Method: In this in vitro study, stem cells were removed from the immature teeth. After cultivation and third passage, metronidazole, ciprofloxacin, minocycline, and clindamycin were placed in the cell culture medium alone , paired, and in combinations as triple antibiotic paste (TAP) (metronidazole, ciprofloxacin, and minocycline) and mTAP (metronidazole, ciprofloxacin, clindamycin) with doses of 25, 50, 100, 200, 400μg/ml. After 1 and 3 days, cell viability in the culture medium was assessed using the MTT method ([4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). SPSS software version 24, descriptive statistics methods, and statistical tests such as Kruskal-Wallis and Mann-Whitney tests were adopted to analyze the data. Results: Analysis of MTT findings indicated that the use of mTAP at 100μg/ml and TAP at 200μg/ml had no adverse cytotoxic effect on stem cells in the first 24 hours, compared to the control group. The cell viability decreased at higher concentrations, although it was not statistically significant. After 72 hours, the toxicity of concentrations higher than 100μg/ml of mTAP and 400 μg/ml of TAP significantly mitigated the percentage of viable cells. Conclusion: The obtained results demonstrated that the concentration of 100 μg/ml of mTAP could replace TAP in regenerative endodontic treatments at the studied time intervals without worrying about the toxicity.


Introduction
Endodontic regenerative methods are biologically based on methods, which are devised to replace damaged tissues such as dentin and root structure and dentin-pulp complex cells [1]. Since the time that the protocol proposed by   [2], several studies have focused on the treatment of immature permanent teeth regarding the clinical efficacy of methods and materials to enhance the regenerative endodontic outcome protocol. Given that the reduction of microbial load in regenerative endodontics is realized by in-channel washing and dressing, the selection of a proper material might be a critical factor for attaining effective cavity disinfection, and the balance between the antimicrobial effect of chemicals and their harmlessness to stem cells is of great importance [3]. In 1996, Hoshino et al. [4] proposed a combination of metronidazole, ciprofloxacin, and minocycline, called triple antibiotic paste (TAP), as an alternative to calcium hydroxide for intracanal dressing in the regenerative process, due to the induction of necrotic tissue by calcium hydroxide. Due to the multimicrobial nature of dental infections, the single use of antibiotics cannot create a medium, free from bacteria in the root canal. As a result, using a combination of antibiotics against all endodontic pathogens is required to hinder the microbial resistance [5].
Both in in-vitro [6] and in clinical trials [7][8], various antibiotic combinations such as TAP have been shown to be highly efficient against common bacteria in the root canal system, Numerous studies have reported that minocycline causes discoloration of teeth [4,[7][8], and many attempts have been made to reduce the discoloration when using TAP [9]. However, this antibiotic is difficult to be obtained in the Iranian market. Unlike minocycline, clindamycin does not discolor deciduous teeth and the permanent teeth replacing them and it is readily available. Furthermore, clindamycin has fewer side effects and drug interactions than erythromycin and amoxicillin, and penetrates well into the most tissues and abscesses, and its long half-life is effective on anaerobic gram-positive and gram-negative bacteria [10].
Casamassimo et al. [11] recommended that minocycline should be replaced with clindamycin in TAP. The new combination was named modified triple antibiotic paste (mTAP). Recent studies have shown successful results using TAP and mTAP in the treatment of deciduous tooth pulp, and the results of antimicrobial tests of both compounds have been quite similar [12]. In addition, Lin et al. [13] showed in a study that regenerative endodontic treatment using other mix paste in immature permanent teeth with apical periodontitis causes disappearance of periapical lesions in all specimens after 12 months of follow-up. The cytotoxic effect of TAP on primary pulp stem cells of deciduous teeth has also been reported [14]. There are concerns about the destructive effect of mTAP on the stem cells from the apical papilla (SCAPs) of immature permanent teeth. Therefore, this study aims to investigate the cytotoxicity of the mTAP complex on the SCAPs of permanent human teeth in comparison with TAP.

Sample selection
After obtaining informed consent from the patients, four

Preparation of antibiotics
First, the necessary medications, including metronidazole (5g), ciprofloxacin (5g), minocycline (25mg) and clindamycin (1g) (Sigma Aldrich; Germany), were pre-  Table 1  When applying ciprofloxacin, a concentration of 400 μg/ml of ciprofloxacin significantly reduced the cell viability in the first 24 hours (compared to the control group) 12 times more than 200μg/ml. With clindamycin, after 24 hours, a concentration of 400μg/ml led to a significant 80% reduction in cell survival, compared to 100 μg/ml, and after 72 hours, up to a concentration of 200 μg/ml, the cell survival rate was higher, but at a concentration of 400 μg/ml, a significant reduction in cell survival was observed. After 72 hours, minocycline increased cell survival by increasing the antibiotic concentration, which was statistically significant at 100, 200, and 400μg/ml. The TAP combination did not exhibit an apparent cytotoxic effect in the first 24 hours up to the concentration of g 200 µg/ml, compared to the control group, but at the concentration of 400 µg/ml, the percentage of cell viability decreased by 50% compared to the concentration of 200 µg/ml. In addition, after 72 hours, the concentration of 100 µg/ml showed a 40% reduction in cell viability compared to 50 µg/ml. There is a statistically significant difference in the 400 µg/ml concentration of TAP after 72 hours (Table 1).

Figures 2 and 3 and
In the case of the mTAP combination, the concentration of 100 µg/ml showed a 14% reduction compared to 200 µg/ml for cell survival during the first 24 hours, and 50% of the cells were destroyed at a concentration of 400 µg/ml compared to the control group. After 72 hours, cell growth increased up to the concentration of 100 µg/ml, but at the concentration of 400 µg/ml, cell survival percentage undergone a negative trend, and the highest cytotoxicity was observed, which was statistically significant ( Table 1).
The Kruskal-Wallis test results showed that the difference between the mean cell viability in the groups of single, binary, and ternary antibiotics at different doses  is less toxic than mTAP (p <0.05).

Discussion
Endodontic treatment of immature teeth due to caries or trauma has been reviewed since the release of regenerative endodontic procedures. Important studies have been conducted in this field to evaluate the antimicrobial effect and biocompatibility of intracanal medications in regenerative endodontics [14,[17][18][19][20]. Due to the essential need for the survival of SCAPs to promote hard tissue deposition, the present study was performed to determine the toxicity of the two antibiotic combina-tions, TAP, and mTAP, on SCAPs of permanent human teeth. One of the substances whose benefits in treating pulp diseases of immature permanent teeth have recently been proven is TAP [21][22][23]. Recent findings by Karczewski et al.'s study [24] suggest using clindamycinmodified antibiotic polymer (polydioxanone) nanofibers as a reliable therapeutic alternative for pastes containing minocycline.
Recently, a cytotoxic effect of TAP on SCAPs at the 50μg/ml concentration has been observed, but such effect was absent at the concentration of 10 μg/ml after 3 and 5 days [25]. This finding contradicts the findings of the present study. The contradiction may be due to differences in the time intervals used in studies. Although this difference in results may be due to differences in the cells types or manufacturers of the antibiotics acquired by the researchers, clinicians should be aware that antibiotics from different sources can have varying  [14,17,27]. Since low doses of antibiotics have been used to make antibiotic-releasing scaffolds [28][29][30][31][32][33] and due to the suitability of the slow release of low-dose drugs from the scaffold, the study of the toxicity of low-dose antibiotics on different types of stem cells, as well as their antimicrobial effects against different types of bacteria that may help in the construction of scaffolds is essential.
One the important finding of the present research is the similar toxicity effect of TAP and mTAP at a 100 μg/mL concentration at the studied time intervals on SCAPs. In this study, cell growth of SCAPs in binary combinations of antibiotics at low concentrations (25 and 50 μg/mL) was also observed. These findings may show similar behavior of antibiotics at lower concentrations while having different effects at higher concentrations. For the future studies, it is recommended to evaluate the effect of TAP and mTAP at different concentrations at times longer than 72 hours. According to the preliminary results, it seems that the proliferation of apical papilla stem cells can neutralize the primary cytotoxic effect.

Conclusion
Apical papillary mesenchymal stem cells are present at the root end of immature teeth. The presence of mTAP at a concentration of 100μg/mL can replace TAP at the studied time intervals in regenerative endodontic treatments without any concern about the toxicity.